The goal of this proposal is to develop a fermentative strategy for the large-scale production of L-fucose and other rare sugars. L-fucose (6-deoxy-L-galactose) is an important hexose deoxysugar found in a variety of organisms attached to an array of macromolecules. These L-fucose containing glycans exhibit a wide range of medicinal properties including supporting infant health (L-fucose containing human milk oligosaccharides); anticoagulant and antithrombotic, antivirus, antitumor, anticancer and immunomodulatory, anti-inflammatory, blood lipids reducing, antioxidant, activitiy against hepatopathy, uropathy and renalpathy, gastric protective effects and therapeutic potential in surgery (L-fucose containing polymers). The L-fucose monomer has therapeutic properties such as inhibiting virulence factors and is also an invaluable synthetic starting material for a wide range of molecules including human milk oligosaccharides, blood group antigens, E and P-selectin antagonists, and functionalized L-fucose derivatives. In addition to pharmaceutical relevance, L-fucose also possesses topical properties attractive to the cosmetic industry including anti-aging, wrinkle reducing and is safe for sensitive skin. Despite the impressive range of bioactivity discovered thus far, L-fucose remains prohibitively expensive and unavailable in the scale needed to support these applications. We feel a fermentative approach is needed to meet these large-scale requirements and to provide the glycoresearch community with this important building block needed to prepare scarce or unavailable glycans. A key aspect of our strategy is the production of an engineered L-fuculose-1-phosphate aldolase that no longer requires the phosphorylated donor substrate, dihydroxyacetone phosphate. Instead, the novel L-fuculose-1-phosphate aldolase (FucA) will catalyze the condensation between dihydroxyacetone and L-lactaldehyde to form L-fuculose, thereby bypassing the typical sugar-phosphate intermediate. Through metabolic engineering, we envision an E. coli system capable of integrating this engineered L-fuculose-1-phosphate for the specific synthesis of L-fucose. In Phase I we will demonstrate the feasibility of a fermentative L-fucose system by identifying and engineering a mutant FucA enzyme capable of condensing dihydroxyacetone and L-lactaldehyde to form L-fuculose for the ultimate production of L-fucose. In Phase II we will focus on the genetic and metabolic engineering of E. coli for the production and accumulation of the substrates needed for the engineered L-fuculose-1-phospahte aldolase. We propose a yield of 50-100 g/L production of L-fucose after integration of the engineered L-fuculose-1-phosphate aldolase and subsequent optimizations. In Phase III we will commercialize L-fucose as well as other rare sugars produced by the engineered L-fuculose-1-phosphate aldolase using various acceptor aldehyde substrates.